Mechanistic advances in osteoporosis and anti‐osteoporosis therapies

Abstract Osteoporosis is a type of bone loss disease characterized by a reduction in bone mass and microarchitectural deterioration of bone tissue. With the intensification of global aging, this disease is now regarded as one of the major public health problems that often leads to unbearable pain, risk of bone fractures, and even death, causing an enormous burden at both the human and socioeconomic layers. Classic anti‐osteoporosis pharmacological options include anti‐resorptive and anabolic agents, whose ability to improve bone mineral density and resist bone fracture is being gradually confirmed. However, long‐term or high‐frequency use of these drugs may bring some side effects and adverse reactions. Therefore, an increasing number of studies are devoted to finding new pathogenesis or potential therapeutic targets of osteoporosis, and it is of great importance to comprehensively recognize osteoporosis and develop viable and efficient therapeutic approaches. In this study, we systematically reviewed literatures and clinical evidences to both mechanistically and clinically demonstrate the state‐of‐art advances in osteoporosis. This work will endow readers with the mechanistical advances and clinical knowledge of osteoporosis and furthermore present the most updated anti‐osteoporosis therapies.


F I G U R E 1
The overview of etiologies and characteristics of osteoporosis. Genetic factors, hormone deficiency and senescence, etc., can lead to the dysfunction of osteoblasts and osteoclasts thus causing osteoporosis. Osteoporosis can increase the risk of fractures in different body parts, along with changes in serological indicators and imaging parameters. BMD, bone mineral density.
With the increasing pressure of global aging population, osteoporosis may become a new worldwide challenge, causing an enormous burden at both the human and socioeconomic layer. Statistics show that nearly one-third of women and one-fifth of men in the world are suffering or will suffer from osteoporosis. 1,2 The complete structure and sound function of bone tissue depend on the bone homeostasis maintained by osteoblasts and osteoclasts. After reaching the peak bone mass before age 35 years, calcium, phosphorus, and other matrix elements in the skeleton will be lost to different degrees. 3,4 The imbalance of bone remodeling continues to accumulate, resulting in the reduction of bone mass and microarchitectural deterioration of bone tissue, ultimately leading to osteoporosis. Menopause and senescence affect the balance between osteoclasts and osteoblasts in different ways, thus presenting different characteristics of bone turnover rate.
Classic anti-osteoporosis therapies include antiresorptive and anabolic agents. Although a large number of drugs have been developed to prevent fractures, many patients still do not receive adequate treatment due to poor compliance and large side effects. 5 In this study, we divided anti-osteoporosis therapy into three categories: small molecular interventions, monoclonal antibodies, and synthetic peptides, according to the source, mode of action, and characteristics of drugs. Starting from the etiology and pathogenesis of primary osteoporosis, we systematically reviewed new therapeutic strategies or new clinical evidence on the basis of existing anti-osteoporosis therapies, hoping to endow readers with the mechanistical advances and clinical knowledge of osteoporosis.

Genetic factors
Osteoporosis has strong genetic susceptibility. Family studies show that a significant amount of the variance in peak bone mass is genetically determined. 6 Twins Study revealed that genetic factors account for 25%-45% of the variation in age-related bone loss. 7 Genome-wide association studies have identified over 200 osteoporosis susceptibility loci, including COL1A1, COLIAI2, LRP5, SOST, etc., but the clear structure, number, distribution, and size remain largely unknown. 8 Among them, DAAM2 used to be predicted to play a role in canonical Wnt signaling. 9 Further study showed that in the femur of mice with Daam2 knockdown, the porosity of cortical bone increases and the structure of bone trabecula is impaired, resulting in a significant decrease in bone strength. 10

Endocrine factors
A number of hormones contribute to bone metabolism, including estrogen, testosterone, calcitonin, and parathyroid hormone (PTH). Changes in estrogen levels in women during pregnancy or after menopause can lead to bone loss and risk of fractures. 11,12 Similarly, severe gonadal steroid deficiency induces bone loss in adult men, but the impact of testosterone level change is relatively small. 13 PTH has a two-way effect on bone regulation. Chronic deficiency of PTH in patients with hypoparathyroidism can lead to abnormality of bone microstructure, reduction in bone remodeling, and increased risk of vertebral fracture. 14,15 In hyperparathyroidism, continuous exposure to high levels of PTH causes increased bone absorption, and even mild symptoms can reduce bone mineral density (BMD) and cause fragility fractures. 16 Although the function of calcitonin is weak in physiological state and there is no correlation between basal calcitonin level or calcitonin reserve and change in BMD, 17 calcitonin together with PTH can maintain normal calcium and phosphorus metabolism, which is vital for maintaining bone mass. 18 Meanwhile, excessive glucocorticoids due to self-reason (hypercortisolism) or long-term medication can increase the risk of vertebral fractures and bone loss. 18,19 However, the dose and severity of glucocorticoids are not positively correlated. Similarly, hypermetabolism caused by hyperthyroidism can also lead to osteoporosis. 20

Senescence
Osteoporosis is one of the most common diseases associated with senescence. More than half of adults over 50 years old suffer from osteoporosis in the USA. 2 One of the main causes of SOP is a decrease in hormone levels. Agerelated inactivation of hormone-binding globulin can lead to testosterone and estrogen inactivation. 21 A further consequence of senescence is a reduction in osteoblast and osteoclast synthesis and secretion ability, which will slow down the rate of bone reconstruction.

Nutritional status
Calcium, phosphorus, and magnesium are the main elements of bone, and the deficiency or proportion change of these elements may lead to bone synthesis disorder and increased bone loss. Adequate calcium and dietary protein intake can reduce fracture risk. 22 Conversely, insufficient nutritional calcium and vitamin D can cause rickets or osteomalacia in children or adolescents and osteoporosis in adults. 23 Calcium supplements to maintain calcium balance have now been regarded as a standardized basic means to prevent and treat osteoporosis.

Lifestyle
Smoking has been identified as an explicit risk factor for osteoporosis. Smoking is associated with reduced bone mass and increased bone loss, while quitting smoking has been clearly shown to improve BMD and reduce fracture risk. 24 The effect of alcohol on bone depends on the dosage. Although light (less than 15 g/day) to moderate (less than 30 g/day) drinking generally lowers the risk of fractures, 25 consuming more alcohol will lead to a continuous increase in osteoporotic fracture risk. 26 A prospective study suggests that sarcopenia has a higher risk of developing osteoporosis. 27 Moderate physical activity can exert mechanical stimulation on bones and strengthen muscle function, which is considered as a bone protection factor. Mechanical loading above daily activ-ity can improve BMD. 28 Many studies have demonstrated that exercise can improve physical function, enhance the quality of life, and reduce pain in PMOP patients. 29 A meta-analysis showed that different kinds of exercise have various effects on the BMD of different parts of skeleton. 30 Being aware of etiologies of osteoporosis can help clinicians prevent the occurrence of osteoporosis, and further insights in the molecular mechanisms contribute to the development of both prevention and intervention treatments. As a result, we will have a brief review of advances in molecular mechanisms of osteoporosis to benefit future clinical work.

PATHOGENESIS OF PMOP
Approximately half of women will once have a fracture after the age of 50 years due to the decrease in estrogen. 31 In fact, women have higher rates of osteoporosis than men at any age because of estrogen deficiency. 32 Estrogen has a significant impact on maintaining the homeostasis of the endocrine system, cardiovascular system, metabolic system, and bone development. Estrogen deficiency induces bone loss in both cancellous and cortical. 33 Ovarian aging results in a higher risk of osteoporosis in women. 34,35 Even in men, estrogen remains the major regulator of the bone. 36 Having precise knowledge on the role of estrogen in bone metabolism is the foundation of managing PMOP. Estrogen acts in a "two-step" way. First, estrogen binds to the estrogen receptors (ERs) in the cytoplasm, and the ERs then dimerize and translocate to the nucleus. 37 Estrogen passively passes through the cell and nuclear membranes and then combines with the receptor. The complex of ligand and receptor binds to specific sequences in the regulatory region of the target genes, and these sequences of DNA are known as estrogen response elements (EREs). 38 There are two types of ERs, alpha (ERα) and beta (ERβ). They have different affinities to estrogen and different distributions 39 (Figure 2). ERs belong to the steroid/thyroid hormone superfamily of nuclear receptors. All members are composed of three functional domains, which are independent of each other but interact. The DNA-binding domains are different between ERα and ERβ. Besides, the ligand-binding sites of ERα and ERβ have slight differences, which enables the development of selective estrogen receptor modulators (SERMs) 40 ( Figure 2). Postmenopause can affect estrogen by decreasing the level and changing the concentration of different estrogen. There are three major forms of physiological estrogens in females: estrone (E1), estradiol (E2 or 17β-estradiol), and F I G U R E 2 The mechanism of selective estrogen receptor modulators (SERMs) on bone homeostasis. The main estrogen receptor types (ERα or ERβ) distributed in different tissues and organs are different. SERMs can selectively activate ERs in the skeleton after oral administration, regulate osteoblasts, osteoclasts, and osteocytes, and upregulate the ratio of osteoprotegerin (OPG) to receptor activator of nuclear kappa-B ligand (RANKL). estriol (E3). In postmenopause, serum E2 levels decrease by 85%-90% and serum E1 levels decrease by 65%-75% from mean premenopausal levels. 41 The decline in estrogen after menopause can affect bone mass in many ways, and we will introduce these effects, which may become a guidance of medical choices. 41 The long list of possible targets of ER includes cytokines, such as interleukin (IL)-1, 42,43 IL-17, 44,45 IL-6, 43,46 IL-7, [47][48][49] and tumor necrosis factor-alpha (TNF-α) 43,50 (Table 1). Cells such as T and B lymphocytes, macrophages, and dendritic cells are affected by estrogen, too. 51

Estrogen deficiency directly related to osteoblast and osteoclast activity
Receptor activator of nuclear factor-kappa-b ligand (RANKL) is the crucial molecule needed for osteoclast development from myeloid precursors. Macrophage colony-stimulating factor (M-CSF)/RANKL signaling stimulates absorption activity. Estrogen blocks RANKL/M-CSF-induced activator protein-1-dependent transcription, thus restraining RANKL-induced osteoclast differentiation. 52 Estrogen inhibits RANKL-stimulated osteoclastic differentiation of monocytes by inducing ERα binding to BCAR1, a scaffolding protein. 57 However, mechanisms by which estrogen interacts with osteoclasts via RANKL signaling remain ambiguous. Other mechanisms have also been proposed. Binding of estrogen and ERα leads to osteoclast apoptosis via Fas/FasL system-mediated apoptotic induction. 53 Comparing protective efficiency in LYN (a kind of intracellular membrane-associated protein tyrosine kinase, which has a crucial function in signaling intermediaries) knockdown osteoclasts and control conditions shows the importance of LYN as a key mediator of the effect of estrogen on osteoclastogenesis. 58 There are reports about osteoclast-specific HIF1α inactivation antagonizes estrogen-deficient bone loss, and the HIF1α was destabilized by estrogen treatment. 59 Thus, HIF1α represents a promising therapeutic target in osteoporosis.
Furthermore, estrogen has an effect on osteoblasts. Estrogen can help prolong the survival of osteoblasts. Estrogen reduced apoptosis in differentiating osteoblasts by promoting autophagy, thus contributing to their longer lifespan. 54 Estrogen has been shown to protect osteoblasts from apoptosis, and the protective effect may be mediated by heat shock protein 27. 60 Estrogen can prompt the synthesis function of osteoblasts. Estrogen upregulates BMP-4-induced Smad1/5/8 phosphorylation in osteoblasts, and the effects can be reversed by the presence of ER antagonists. 55 In addition, ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing the proliferation and differentiation of periosteal cells. 61 Estrogen deficiency is not restricted to osteoclasts and bone resorption but also affects bone matrix composition and response of osteoblasts to mechanical stimulation. 62 There are reports about estrogen augments shear stress responsiveness of signal transduction and gene expression in bone cells via ER-mediated increases in β1-integrin expression. 56 TA B L E 1 List of pathogeneses of postmenopausal osteoporosis (PMOP) and approaches these pathogeneses affect osteoblasts and osteoclasts.

Pathogenesis of PMOP Ways to have impacts Influences on osteoblasts or osteoclasts References
Inflammation due to estrogen deficiency Interleukin-1 Increasing osteoclasts formation 42 Promoting osteoclasts survival 43 Interleukin-17 Stimulating osteoblast and osteoclasts differentiation 44 Stimulating expression of RANK and receptor of M-CSF 45 Interleukin-6 Indirectly stimulating osteoclasts activity via stimulating expression of interleukin-1 43 Stimulating osteoclasts differentiation in a RANKL-independent way 46 Interleukin-7 Indirectly stimulating osteoclasts activity via stimulating expression of TNF-α and RANKL 47,48 Inhibiting osteoblastic bone formation. 49 TNF-α Stimulating RANKL expressing 43 Recruiting osteoclasts Stimulating osteoclasts differentiation in a RANKL-independent way 50 Estrogen deficiency RANKL and M-CSF Inhibiting osteoclasts differentiation 52 Fas/FasL system Leading to osteoclasts apoptosis 53 Promoting autophagy Prolonging the survival of osteoblasts 54 Upregulating BMP-4 Prompting the synthesis function of osteoblasts 55 Increasing in β1-integrin expression Augmenting shear stress responsiveness 56 Abbreviations: RANK, receptor activator of nuclear kappa-B; RANKL, RANK ligand; TNF-α, tumor necrosis factor-alpha.

Estrogen deficiency indirectly related to osteoblast and osteoclast activity
Estrogen also indirectly regulates osteoclasts. 44,63 Postmenopausal women often display a chronic low-grade inflammatory phenotype with altered cytokine expression and immune cell profile. 44 Estrogen deficiency in humans elevates the expression of RANKL in lymphocytes. 63 B lymphocytopenia can be regarded as a characteristic of osteopetrosis, suggesting that B lymphopoiesis is regulated by osteoclast activity. 64 CD22, shp-1, bcl-2, and vcam-1 have been upregulated by estrogen via activating ERα and ERβ in B cells, and overexpression of CD22 and SHP-1 in B cells decreases B-cell receptor signaling. 65 Factors affecting B-cell function also affect osteoclasts and osteocytes, indicating a regulatory relationship between B lymphopoiesis, osteoclastogenesis, and osteoblastogenesis. Transcription factors required for B-cell differentiation have unpredicted, pronounced, and non-overlapping effects on osteoblast and osteoclast development. 66 Human estrogen deficiency expands RANKLexpressing T and B cells. Besides, T-cell-produced TNF-α in postmenopause leads to bone loss. 67 Loss of estrogen leads to inflammation that promotes osteoporo-sis. T cells and proinflammatory cytokines are closely connected with estrogen loss. E2 loss leads to chronic low-grade production of the proinflammatory cytokines TNFα and IL-17, both of which are important for osteoclast differentiation. 68 Anti-IL-17 antibody preserved cortical bone parameters, bone biomechanical properties, and histomorphometry in animal models. 69 TNFα can induce osteoclast differentiation RANKL-independent manner, although limited. It also has a synergic effect on RANKL-stimulated osteoclastogenesis. 50,70 Proinflammatory cytokine IL-1 causes activation of the inducible nitric oxide pathway in bone cells and induces bone loss. Recently, the cytokine IL-6 has joined the form of cytokines as a bone-reactive agent. 71,72 There have been reports identifying IL-1β-induced osteoclasts as a contributor to bone erosion in arthritis. 73 At the same time, these inflammatory cytokines also have an effect on inflammatory cells, so the effect of estrogen is not a single line but a complex network. We list the cytokines that are regulated by estrogen and have effects on bone metabolism in order to have a clearer understanding of how estrogen regulates bone metabolism (Table 1).
Reactive oxygen species greatly influence the generation and survival of osteoclasts, osteoblasts, and osteocytes.
Loss of estrogen or androgen decreases the defense against oxidative stress in bone, which explains the decrease in bone matrix associated with the acute loss of these hormones to some extent. 74

Other influences of estrogen deficiency on bone metabolism
Estrogen can also have crosstalk with other hormones, which have effects on bone. Estrogen promotes calcitonin secretion and inhibits bone absorption, enhances the activity of hepatic 25-hydroxylase and renal 1 alpha-hydroxylase, increases the expression level of 1,25-(OH)2D3, and promotes intestinal calcium absorption. 75 In vitro culture experiments demonstrate that E2 and progesterone can stimulate PTH secretion by rapid, direct, and specific effects on parathyroid cells, which implies that in vivo PTH can also be affected by estrogen and disturbed in the postmenopausal period. 76 An integrated 16S rRNA gene sequencing and liquid chromatography-mass spectrometry-based metabolomics approach shed light on the possible relationship between BMD and gut microbiota/metabolite alterations in PMOP. 77

PATHOGENESIS OF SOP
Senescence is involved in changes in biological phenomena of osteoblasts and osteocytes, lack of exercise, and nutrition intake. All of them disturb bone turnover, and we will review how senescence influences bone metabolism. SOP and PMOP are associated intimately but still have some differences.

Senescence in stem cells
Senescence is a complex process associated with various structural, functional, and metabolic changes. At the biological level, senescence results from the impact of the accumulation of a wide variety of molecular and cellular damage over time.
Mesenchymal stem cells (MSCs) are multipotential cells that can self-renew and differentiate into various cell types, including osteogenic and adipogenic fates. 78 Bone marrow mesenchymal stem cells (BMSCs) exhibit an age-related lineage transformation from osteogenic to adipogenic fates, contributing to bone loss. 79 As a result, the senescence of BMSCs directly influences skeletal formation by inhibiting osteogenesis. Human MSCs respond with a set of senescence programs to different stresses, including oxidative stress, damage stress, and chemical agents. The senescence programs of BMSCs include telomere dysfunction, DNA damage, irregular chromatin organization, and strong mitogenic pathway stimulation. 80 When senescence-related stimulation acts on stem cells, senescence-related phenotypes are prompted by the retinoblastoma protein or p53 pathways. Then, the pathways activate the cyclin-dependent kinase inhibitors p16 and p21, which are regarded as markers of senescence. It is worth noting that the pathways can interact with each other to induce senescence. [80][81][82] Other pathways that mediate BMSC senescence are excavated. Researchers have found decreased autophagy in aged BMSCs compared with young BMSCs, and activation of autophagy could partially reverse the BMSC aging. 83 This finding can be a potential therapy to oppose SOP. Depletion of SIRT3 leads to compromised nuclear integrity, loss of heterochromatin, and accelerated senescence in MSCs. The reintroduction of SIRT3 rescues the disorganized heterochromatin and the senescence phenotypes. 84 In vitro research found an interaction between fibroblast growth factor 21 and senescence of MSCs, depletion of FGF21 enhanced the senescence of early-passage MSCs, and overexpression of FGF21 in aged MSCs inhibited senescence. 85 More than researches about alternations in phenotypes, mechanisms about functional influences of senescence have also been developed. 86,87 Senescence has an impact on BMSCs' self-renewal and osteogenic and intercellular communication. 88,89 FOXP1 expression level progressively declines with age. Conditional depletion of FOXP1 in BMSCs leads to premature aging characteristics and impairs MSC selfrenewal capacity in mice. 90 As the common progenitors of adipocytes and osteoblasts, MSCs are delicately balanced; otherwise, the differentiation of osteoblasts and adipocytes will be tipped. Numerous in vitro studies have demonstrated that, although not strict, almost all adipogenic factors inhibit osteogenesis, and conversely, osteogenetic factors restrain adipogenesis. 91,92 Aside from familiar fatedetermining elements of osteogenesis, such as transforming growth factor-β (TGFβ)/ bone morphogenetic proteins (BMPs, members included in TGFβ family), winglessintegrated (WNT) signaling, etc. Long noncoding RNA (LncRNA) and extracellular vesicles (EVs) can also regulate MSCs' fates during skeletal aging. 79 How LncRNAs affect MSC is not precisely understood, but some studies have revealed some candidates of LncRNAs. For example, LncRNA-MEG3 can upregulate the osteogenetic differentiation of MSCs. The changes between the secretomes of senescent BMSCs and young BMSCs may also lead to osteoporosis. Treatment with EVs from MSCs generated from human embryonic stem cells reduces senescence in vitro and in vivo. 93 EVs from other stem cells can also rescue BMSC senescence. The umbilical cord-produced MSC-EVs contain abundant anti-aging signals and rejuvenate senescing adult bone marrow-derived MSCs. 94 The functions of MSCs are not limited to their immense differentiation potential; they have a significant capacity in immunoregulation by inhibiting the immune response and exerting a function generally known as immune adjustment. 80,95 MSCs, can alter the frequency and function of memory lymphocytes, including Th17, 96 follicular helper T (Tfh) cells, 97 and gamma delta (γδ) T cells. 98,99 Dysregulation of T cells due to MSC senescence may be related to dysfunctions in bone metabolism, and more related research is needed in this territory. 100,101 Different lineages from MSCs have interactions, for example, osteolineage cells are believed to be a population linked to the regulation of hematopoietic stem cells. 102 In an aged individual, both niches are interfered and this may contribute to osteoporosis.
Explorations in MSCs' senescence modulation still cannot meet the need for clinical use, and there remain conundrums in inhibiting senescence in MSCs and clinical transformation.

Senescence in osteoblasts and osteoclasts
Age-related osteoblast dysfunction related to changes in bone microenvironment and their own senescence. The age-related impairment in MSCs leads to an impact on osteoblastic cell proliferation, and the underlying mechanisms are discussed above. However, the senescence impacts lifespan of osteoblasts. Enhancement of P53 leads to bone loss in mice. Likewise, genetic depletion of P53 will accumulate bone mass due to enhanced proliferation and reduced apoptosis. 103,104 The differentiation and function of osteoblasts are impaired. In rats, accumulation of preosteoblastic cells shows up with decreased number of mature osteoblasts with increasing age, suggesting that impaired osteoblast differentiation is a potential mechanism for age-related impaired bone formation. 105 A more recent in vitro study demonstrated that senescent cell conditioned medium disturbs osteoblast mineralization and enhances osteoclast progenitor survival. 106

Hormone deficiency
Several hormones may play essential roles in bone homeostasis, including estrogen, testosterone, cortisol, PTH, and thyroid hormones. The inharmony in these hormones will impact the concentration of calcium/phosphate and bone homeostasis. 107,108 The impacts of estrogen deficiency have been discussed above and we specifically do not repeat here.
Parathyroid and thyroid hormones are both important systematic bone-remolding modulators. Thyrotoxicosis is well known to cause severe osteoporosis and fracture. Subclinical thyroid disease will lead to bone loss and osteoporosis. Thyroid hormone can act in the skeleton directly by access to thyroid receptors. 109 Significant and physiologic changes in thyroid parameters are observed during aging, and whether the changes tip the skeletalmetabolism balance related to thyroid remains to be observed. 110 The mechanisms by which PTH prompts osteoblast differentiation have not been thoroughly studied. However, in vitro studies have demonstrated that PTH induces osteoblast differentiation mainly via activation of the Wnt/β-catenin pathway in osteoblastic MC3T3-E1 cells. 111 Intermittent PTH promotes osteoblast differentiation, in part, by its ability to promote exit from the cell cycle, to activate Wnt signaling in osteoblasts, and to inhibit the Wnt antagonist sclerostin in osteocytes. 112 However, hypoparathyroidism is more prevalent in the younger population, which may related to surgery for thyroid disorders. 113 But the studies in aging rats have revealed declines in PTH regulation of signal transduction pathways, which might relate to declines in the number of receptors. 114

Exercise
Lack of exercise in old age can weaken bone and muscle parameters not limited to locomotor limbs but systematically. 115 Local mechanical environment, such as bone strain, fluid shear flow, and electromagnetic fields within the bone influences osteoclasts convene to a particular location. 116 The underlying theories have not been clear yet. But many studies have revealed the role of osteocytes in mechanical load bone remodeling. At the cellular level, mechanical load influences fluid flow in the osteocyte microenvironment. Investigating the molecular mechanisms of osteocyte mechanical-biological conversion is an urgent need. 117 The finding of Piezo1, a mechanosensitive ion channel, explains the mechanism to some extent. 118 Mechanical loading signals can transduce from osteocytes to osteoblasts via gap junction protein, Connexin43. 119 Using the tooth-movement model, researchers find that mechanical load elevates tooth movement and prompts the number of M1-like macrophages. 120 Mechanical loading can influence bone metabolism by having effects on osteocytes, osteoblasts, and osteoclasts.
Muscles and bones are considered as functional unities, and the degeneration in muscles will lead to bone loss.
The potential molecular mechanisms are not limited in mechanical coupling theory but the secretory nature of bones and muscles can also affect each other. The secretory proteins included insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), IL-6, IL-15, myostatin, osteoglycin, family with sequence similarity 5,member C (FAM5C), Tmem119, and osteoactivin. 121 Exercise also has an effect on endocrine. Exercise can influence serum PTH. Impact exercise training lowers the serum basal PTH levels and possibly enables greater difference between the basal PTH and transient exerciseinduced PTH peaks which lead to osteogenic effects. 122 A 40-min downhill exercise can prevent or mitigate PMOP in sedentary women by avoiding circadian PTH oversecretion. 123

PATHOGENESIS OF IOP
In a study of the Mayo Clinic screening individuals aged 20-44 years, the incidence of IOP in this age group was only 0.4 cases per 100,000 person-years, with no difference between genders. The cause of IOP is believed to be connected with genes. But which genes are related to IOP remains unclear. Researchers are revealing the mystery in causing of IOP with methods of genome-wide association studies. 124 Ferrari et al. 125 reported that LRP5 variants are implicated in idiopathic male osteoporosis. The positivity rate of pathogenic variants is twofold higher in children compared to adults, indicating that the frequency of mutation of related genes may be related to the age of beginning. Using gene panel sequencing in diagnosis of children and young adults referred for IOP reveals that the most frequent mutation happened in LRP5, WNT1, and COL1A1 or COL1A2 genes. 126 In the way of exploring correlated pathologic gene mutants in IOP, researchers have also found some gene mutations relevant to bone, but the mechanism still perplexing. 127 In addition, the change in expression of bone metabolic genes also leads to IO. Patsch et al. 128 analyzed the iliac crest biopsies of men with IO and found decreases in the expression of WNT10B, RUNX2, RANKL, and SOST. The report also found positive correlations of WNT10B with RUNX2, osteocalcin, and RANKL, which indicates potential inhibitory downstream effects on osteoblastic transcriptional activity. A set of circulating microRNAs significant have consistently regulated in patients with osteoporosis (including IOP). Among the set of microRNAs, eight miRNAs are excellent resolving devices for patients with low-traumatic fractures, regardless of age and sex. Correlation analysis identified significant correlations between the set miRNA and P1NP (a bone turnover marker, indicating bone formation), iPTH, TRAP5b (tartrate-resistant acid phosphatase 5b, a marker of bone resorption), osteocalcin, as well as BMD. 129 Some of them have been demonstrated to be associated with bone microstructure and histomorphometry. 130 However, these studies suggest that the system expression change of related genes can be the cause of IOP. With the knowledge about IOP going deep, IOP and nephrolithiasis are believed to be highly connected. Some researchers consider idiopathic nephrolithiasis and osteoporosis as two possible manifestations of a unique clinical syndrome. 131,132 Most of the pathogenic factors affect bone by affecting the differentiation, survival and function of osteoblasts and osteoclasts. In addition, these pathogenic factors interact with each other and form a complex etiologic network, which makes the treatment of osteoporosis greatly complicated.

ANTI-OSTEOPOROSIS THERAPIES
Classic anti-osteoporosis therapies include anti-resorptive and anabolic agents, whose mechanisms are being clarified gradually. Based on the source, mode of action and characteristics of drugs, we divided anti-osteoporosis therapy into three categories in this review: small molecular interventions, monoclonal antibodies, and synthetic peptides. Among them, the use of small molecule interventions has the longest history. Before the precise mechanisms underlying its action were elucidated, inorganic small molecules such as bisphosphate and strontium salts have entered the clinic. Meanwhile, more and more organic small molecular inhibitors and monoclonal antibodies based on molecular targets have been developed. Some of which have obtained sufficient clinical evidence. Besides, small molecular nutrition supplements such as vitamin D and vitamin K are recommended as effective methods to enhance BMD and reduce fracture risk. Synthetic peptides based on natural hormone structure have higher yield, stronger physiological activity, and lower immunogenicity and can be personalized modified. The available synthetic peptides, such as teriparatide and abaloparatide, have unique anabolic effects, but due to the multiple targets, their safety still needs to be further evaluated.

Bisphosphonates
Since it was found to inhibit bone resorption in the 1960s, bisphosphonates (Bps) have become the first-line agents used to treat osteolytic diseases, including PMOP. 133 Bps contain a core skeleton of P-C-P bonds and can stably complex with Ca in hydroxyapatit, leading to the ability to rapidly clear from circulation and selectively target bone 134 (Figure 3). Bps deposited on the bone surface then act on mature osteoclasts through endocytosis during bone resorption. 135 The side chain is the main functional area for Bps, which determines the pharmacological activity and anti-resorptive potency. The first-generation Bps contain simple short side chains, which can replace the β-γ phosphoric acid group of adenosine triphosphate (ATP), producing non-hydrolyzable ATP analog (AppCp-type analogs of ATP) and playing a role by inhibiting ATP-dependent intracellular enzymes. 136 At present, nitrogen-containing Bps with complex side chains are widely used. Nitrogen-containing Bps can inhibit farnesyl pyrophosphate synthase in the mevalonate metabolic pathway, thus preventing the production of isoprenoid lipids, which is necessary for protein prenylation 137 ( Figure 3). The loss of isoprene will lead to protein dysfunction including small GTPase, thus affecting cell migration, adhesion, polarization, vesicular transport, and membrane ruffling, which is essential for osteoclasts 138 (Figure 3). In addition, the accumulation of upstream metabolites and unprenylated proteins can also produce cytotoxicity, resulting in the induction of osteoclast apoptosis.
Bps can be administered orally or intravenously, which will lead to different side effects. Oral Bps has poor absorption and may cause low compliance due to gastrointestinal reactions. 5 Giving Bps intravenously bypasses the problem of gastrointestinal intolerance, but it will multiply the risk of bisphosphonate-related osteonecrosis of the jaw (BRONJ). A high dosage of Bp is the main cause of BRONJ. In addition, trauma and infection are closely related. 139,140 Unfortunately, statistical results show that no effective interventions for managing BRONJ have been found. 141 Besides, using Bps for a prolonged period has been linked to an increased risk of atypical femoral fractures (AFF), 142 which might be related to the accumulation of microdamage in bone caused by low bone turnover rate 143 and irregular absorption in intermittent administration mode. 144 Taking a drug holiday is an effective way to reduce the side effects after taking Bps for 3-5 years, and the dosage can be adjusted according to the examination results, including T score and BMD. 145,146 Although taking Bps does potentially negatively affect the remodeling of the fracture callus, it will not lead to delayed healing. 147 So, it is still recommended to be used early in osteoporotic fractures. 148 Currently, risedronate and ibandronate are only approved to treat PMOP. Alendronate and zoledronate are also authorized by the Food and Drug Administration (FDA) to increase bone mass in men with osteoporosis and treat glucocorticoid-induced osteoporosis and Paget's disease of bone (a chronic osteomatoid degeneration that can cause bone expansion, deformity and strength reduction).

Strontium salt
Strontium (Sr) is a trace element that mainly exists in skeleton system. Due to the physical and chemical similarity of Sr and Ca, Sr is probably built into the hydroxyapatite crystal to play a role. The mechanism of Sr is related to the delayed activation of calcium sensing receptors. 149 On the one hand, Sr inhibits the maturation, TRAP expression, and hydroxyapatite resorption of osteoclasts. 150 On the other hand, Sr promotes the differentiation and proliferation and collagen and non-collagen protein synthesis of osteoblasts. 151 In addition, Sr can also adjust the crosstalk between osteoblasts and osteoclasts by inhibiting the expression of RANKL. 152 Strontium ranelate (SrR) is composed of two stable divalent strontium ions and one ranelate. Several animal experiments have shown that SrR can accelerate the healing process and improve bone mechanical properties. [153][154][155] So, adding SrR into the tissue engineering scaffold to repair bone defects and promote bone regeneration under osteoporotic conditions have become a potential topical application. Including SrR-incorporated bioceramic scaffolds, 156 composite gelatin coatings containing SrR-carrying halloysite nanotubes. 157 Some randomized controlled trials (RCTs) suggest that the level of bone formation markers increased and bone destruction markers decreased in people taking SR for at least 1 year. 158 However, oral SrR may increase the risk of thromboembolic disease, 159 and SrR was withdrawn in 2020. At present, only strontium succinate is used to treat osteoporosis in Europe, and strontium chloride is indicated to relieve bone pain in patients with painful skeletal metastases.

Calcitriol
Calcitriol is the activated form of vitamin D through hepatic metabolism. Together with PTH, they jointly maintain calcium and phosphorus homeostasis. 160 Calcitriol can upregulate calcium transient receptor potential vanilloid channel 6 and calbindin-D9k in the enterocyte, thus stimulating calcium absorption. 161 Adequate calcium intake is considered to maintain an appropriate blood calcium concentration and prevent the mobilization of bone calcium, thus reducing bone resorption and slowing bone loss. 162 High extracellular calcium can induce osteoblast proliferation 163 and inhibit osteoclasts by regulating RANKL. 164,165 Besides, vitamin D can also directly inhibit osteoclast differentiation, fusion, and bone resorption in a dose-dependent manner. 166 Although the beneficial effect of vitamin D on bone has been confirmed in animals, 167 there is no clinical evidence that substantially higher doses of vitamin D confer any advantage. On the contrary, calcium or vitamin D supplements alone may cause the risk of cardiovascular 168 and renal stones. 169 In an ancillary study of VITAL study, the researchers suggested that taking vitamin D3 alone did not reduce fracture risk in generally healthy midlife and older adults. 170 Similar findings are confirmed in community adults without a known history of vitamin D deficiency, osteoporosis, or fracture. 169 The effect of combined use of vitamin D and calcium to prevent fracture remains to be verified. 171 In Europe, a compound tablet of cholecalciferol and calcium carbonate is used to prevent skeletal-related events in patients with advanced malignancies and the combination treatment of osteoporosis with alendronate, cholecalciferol, and ibandronate. Currently, 400-800 units of vitamin D and 1000-1200 mg of calcium are recommended to be taken together daily, including the total amount obtained from food and supplements. 172,173

Menaquinones
Vitamin K was first found to be associated with bone metabolism in warfarin anticoagulant patients. Menaquinones or vitamin K2 is a carboxylated coenzyme of several vitamin K-dependent proteins, including osteocalcin. 174 Carboxylated osteocalcin can induce calcium deposition on the surface of hydroxyapatite, thus enhancing bone mineralization. 175 While a high level of undercarboxylated osteocalcin in serum is thought to be a risk of osteoporotic fracture in elderly women. 176 A meta-analysis indicated that menaquinone supplementation can maintain BMD and reduce fracture rate in postmenopausal women, which can be an option to counter bone loss. 175,177 However, there is no guideline for the recommended doses and forms of vitamin K in the prevention of osteoporosis. Some scholars believe that 155-188 μg vitamin K should be consumed each day to maintain bone metabolism, while others believe that the carboxylation of osteocalcin requires at least 250 μg vitamin K intake. 178 Since no toxicity data are available, the maximum vitamin K intake is not clearly defined. 179 At present, menaquinones are mainly used to treat coagulation dysfunction and are only approved for the treatment of osteoporosis in several Asian countries, including Japan and China. Vitamin K supplementation from plants and dairy products should be considered as a preventive measure. 180

SERMs
Earlier in the article, we discussed that estrogen deficiency is the leading cause of osteoporosis in postmenopausal women. Estrogen is an important protective factor that can simultaneously regulate osteoblasts and osteoclasts and participate in their crosstalk. Hormone-related therapies using estrogen and progesterone have been shown to reduce estrogen deficiency-mediated increased bone turnover and prevent further bone loss. 181 However, due to risks such as breast cancer exceeding benefits, hormonal replacement therapy is only recommended for the relief of menopausal symptoms in the lowest dose necessary and for the shortest time possible, 181 and estrogen has been replaced by SERMs. SERMs can produce different conformational changes through structural changes and binding with different ERs in different tissues. SERMs act as estrogenic agonists in bone and can antagonize estrogen in breast and uterus, which can effectively reduce estrogen-related risks (Figure 2). To improve the bioavailability and reduce the dosage, a variety of new formulations and administrations have been reported. Examples include an oral raloxifeneloaded bioadhesive nanoparticle made up of Carbopol 940, glyceryl distearate, and D-α-tocopheryl polyethylene glycol 1000 succinate (TGPS), 182 an intranasally gelation raloxifene-loaded chitosan nanoparticles, 183 a raloxifeneloaded nasal delivery misemgel matrix using nanosized self-emulsifying systems, 184 an intravenous human serum albumin-based nanoparticles loaded with raloxifene, 185 a menthol added transdermal formulation containing raloxifene nanoparticles, 186 all of which have yielded demonstrated results in vivo. In addition, some plantbased estrogen-like substances extracted from herbs show similar functions with SERMs. Herba epimedii and its extracted icariin have been widely reported to be able to activate non-genomic ERα signaling selectively and simultaneously affect osteoblasts and BMSCs. 187,188 Wang et al. 189 found a natural product, norlichexanthone, which is an ERα ligand and can exert the therapeutic effect with less estrogen activity.
Currently approved SERMs include tamoxifen, toremifene, raloxifene, ospemifene, bazedoxifene, and lasofoxifene. Tamoxifen and toremifene are first used to treat ER + breast cancer and prevent the breast cancer in high-risk adult women. Both of them are not approved for the treatment of osteoporosis due to their ability to activate endometrial ERs and increase the endometrial cancer risk. 190 Being a partial agonist in the endometrium, ospemifene has positive effects on vaginal dryness and can be used to treat vulvovaginal atrophy. 191 Raloxifene is most widely used in the prevention and treatment of PMOP and has been shown to significantly reduce breaks in the spine but not in the hip fractures in PMOP. Bazedoxifene, together with conjugated estrogens, is used in PMOP at risk of fracture. In Europe, lasofoxifene is approved as a third-generation SERM for osteoporosis with increased fracture risk, but its efficacy and adverse reactions still need further evaluation. 192

RANKL inhibitors
The imbalance of RANKL and osteoprotegerin (OPG) is an essential manifestation of bone homeostasis destruction.
Many studies have proved that targeted RANKL therapy is an effective way to treat osteolytic diseases. In this review, we discuss different methods to inhibit RANKL and RANK binding ( Figure 4). Miyata et al. 193 reported a pyridinylpyrimidine derivative (AS2676293) through high-throughput screening that can rescue rapid bone loss in RANKL-injected mice. This team further developed AS2690168 with a similar structure. In ovariectomized (OVX) mice, AS2690168 can inhibit the decrease in BMD in a dose-dependent manner without affecting the serum osteocalcin level. 194 In addition to osteoporosis, AS2676293 also shows a good therapeutic effect in rats with osteolytic diseases such as fibrous dysplasia 195 and bone metastasis. 196 Melagraki et al. 197 constructed a computer virtual screening system based on structural modeling using known TNF inhibitors. Using this system, Melagraki's team found that T8 and T23 with the function of directly inhibiting TNF and RANKL at the same time and conducted molecular dynamics calculations and in vitro evaluations. After that, they identified another plant-origin small molecule inhibitor, A11. 198 With a large surface area and extended hydrophobic region, A11 shows typical characteristics of effective inhibitors of protein-protein interaction. 198 Jiang et al. 199 synthesized a porphyrin derivative, Y1599. Compared with T8 and T32, Y1599 has higher selectivity for RANKL and can inhibit RANKL-induced bone resorption by downregulating the c-fos/NFATc1 signaling pathway and osteoclast marker genes. Compound Y1599 is further cyclized and oxidized to obtain Y1693. Orally, Y1693 demonstrates good tolerability and efficacy in OVX mice and could also suppress the expression of osteoclast marker genes. 200 In addition, another verteporfin analog shows a dose-dependent inhibition of RANK-RANKL interaction in a competitive ELISA. 201 However, its specificity has not been tested, and there is a lack of in vivo data. 201 SPD304 was first reported to promote the dissociation of RANKL trimer ( Figure 4B), but it is interrupted due to the high toxicity. 202 Rinotas et al. 203 developed several analogs of SPD-304 with improved toxicity profiles, which can selectively inhibit RANKL-induced osteoclastogenesis, without affecting TNF activity or osteoblast differentiation.
In a recent study, Huang et al. 204 identified a binding site on soluble RANKL instead of membrane RANKL and discovered a series of soluble RANKL inhibitors ( Figure 4C). Because T and B cells express membrane RANKL at the same time, 205 targeting soluble RANKL can reduce the side effects of immunity, showing the potential to be superior to antibodies.

Sclerostin inhibitors
At present, several potential sclerostin inhibitors screened by computer virtual screening have been reported, but the functional validation of animal and cell experiments has not been conducted. It includes a small molecular cluster 206 and a group of herbal compounds with an aromatic group, 207 targeting the loop 2 domain in sclerostin. A quinoxaline derivative targeting LRP5/6sclerotin interaction. 208

Anti-RANKL antibodies
Denosumab is the first approved human IgG2 monoclonal antibody against RANKL for treating osteoporosis with high fracture risk ( Figure 4D). A 60 mg dose is administered subcutaneously every 6 months. In the past decade, denosumab has consistently improved BMD and maintained a low rate of fracture risk. 209 However, the effect of denosumab on bone homeostasis is reversible. The bone turnover rate would rapidly rebounds after discontinuation and whether the rebound is proportional to duration is not yet clear. 210 In addition to the common side effects of biological therapy, denosumab also produces rare ONJ and AFF similar to Bps. A 7-year FREEDOM extension indicates that the benefit-risk ratio is 281 for AFF and 40 for ONJ. 211 A retrospective cohort study shows that denosumab was the most common second course of treatment prescribed among patients placed on a drug holiday due to taking Bps first. 212 Conversely, taking SERMs after denosumab is still controversial, and Bps are more recommended to recover BMD loss. 213,214 At present, anti-RANKL antibodies used to treat osteoporosis in phase III clinical trials on ClinicalTrails.gov include MW031 (NCT05215977), LY06006 (NCT05060406), QL1206 (NCT04128163), and CMAB807 (NCT03925051). Besides, some novel modified RANKL variants are reported to induce anti-RANKL immune response as an immunogen, suggesting that immunotherapy can be used to treat osteoporosis. 215,216

Anti-sclerostin antibodies
Romosozumab is the only approved human monoclonal sclerostin antibody and it is used to treat high-risk PMOP patients. Other anti-sclerostin monoclonal antibodies under research include blosozumab (already finished phase II clinical trial), 217 and SHR-1222, 218 a humanized IgG4 monoclonal antibody in a phase I clinical trial. In addition, setrusumab was not developed for osteoporosis when entering the phase III clinical trial. 219 Romosozumab can competitively inhibit the binding of sclerotin to LPR5/6 receptor and prevent proteasomal degradation of β-catenin, thereby ultimately fostering the osteogenic differentiation of MSCs and inhibiting osteoclasts activity to regulate bone mass. 220 However, a few phase III clinical trials indicated that inhibition of sclerostin may elevate cardiovascular risk, which might be related to the cardiovascular protection of sclerotin. 221,222 Meanwhile, the initial anabolic effect of anti-sclerostin treatment may be short-lived and the impact of romosozumab on BMD decreases after continuous use for more than 12 months. 223 This may be because sclerostin inhibition induced an increase in endogenous Wnt antagonist Dickkopf-1 as negative feedback. 224 A bispecific antibody targeting sclerostin and Dickkopf-1 may become a potential therapeutic strategy. 225

Calcitonin
Calcitonin is a natural 32-amino-acid-containing polypeptide secreted by parafollicular cells of the thyroid gland.
Calcitonin controls blood calcium mainly in two ways: inhibiting the differentiation and proliferation of osteoclasts through binding to the specific receptor, and inhibiting the reabsorption of calcium and phosphorus in renal tubules. 226 Recent studies in genetically modified mice suggest that calcitonin appears to inhibit osteoblast activity. 226 Unlike other anti-resorptive agents, calcitonin holds a unique advantage in analgesia and thus could be used to relieve the pain caused by osteoporosis-related vertebral compression fractures. This function might be related to altering Na + channel and serotonin receptor expression or endorphin-mediated mechanisms, but its specific mechanism is not yet clear. 227 The commercialized preparations of calcitonin include recombinant salmon calcitonin and elcatonin, administered intranasally, subcutaneously, or intramuscularly. Traditional calcitonin formulations may cause a sudden drop in blood calcium due to the burst release and require frequent administration because of the short half-life and wide distribution of receptors around the whole body. 228 Li's team continues to report a thermosensitive hydrogel with oxidized calcium alginate and hydroxypropyl chitin added as a versatile platform for sustained release of calcitonin. 229 In this system, calcitonin can be stably released for more than 1 month after a single subcutaneous injection and shows sustained effects in bone trabecula reconstruction in glucocorticoid-induced osteoporosis rats. 230,231 In addition, they also report a bone-seeking hexapeptide-conjugated salmon calcitonin whose femur tissue accumulation is threefold higher in ovariectomized models. 232 Similarly, Wang et al. 233 designed a polylactic acid microsphere coated with tannic acid/PEGylatedsalcatonin layer-by-layer films with unique zero-order release kinetics.
A number of clinical studies suggests that there is a potential risk of malignancy associated with calcitonin use, such as basal cell carcinomas, prostate cancer, and liver cancer. 234 Therefore, calcitonin gets the FDA approval only for the treatment of osteoporotic women at least 5 years postmenopausal without alternative treatments. 235 In Europe, calcitonin is only used for a short term in bone metabolic disorders, including Paget's disease, acute bone loss due to sudden immobilization, and cancerinduced hypercalcemia. 236 Meanwhile, its oral formation (SMC021) was also terminated in the third phase clinical trial (NCT00525798). 237

PTH
PTH is an 84-amino-acid-containing single-chain polypeptide secreted by the main cells of the parathyroid gland. PTH plays a vital role in calcium and phosphorus metabolism and has complex mechanisms on bone metabolism 238 ( Figure 5). In intermittent administration mode, PTH induces the osteogenic differentiation of MSCs and promotes the proliferation and differentiation of osteoblastic lineages. 239 During continuous high-dose administration, PTH indirectly regulates osteoclasts by mediating through RANKL F I G U R E 5 Parathyroid hormone (PTH) bidirectionally modulates bone metabolism. PTH and PTH-related peptides play a role by combining with PTH1R in the skeleton system. Different means of administration will lead to different effects on bone tissue cells, thus producing osteoclastic or osteogenic effects. RANKL, receptor activator of nuclear kappa-B ligand.
secretion from osteoblasts. 240 At present, the synthetic PTH includes teriparatide and abaloparatide, consisting of the first 34 amino acids of PTH and PTH-related peptide (PTHrP), respectively. Unlike teriparatide, abaloparatide binds more tightly to PTH1R RG conformations, leading to a shorter cyclic adenosine monophosphate (cAMP) response. 241 Therefore, abaloparatide causes less bone resorption than teriparatide. 241 Recent studies in rats have also shown that abaloparatide can promote bone formation without increasing bone resorption. 242 In addition, abaloparatide also shows greater osteogenic effects and better tolerance. 243 Both PTH and PTHrP could cause osteosarcoma in a dose-dependent effect when administered in high doses for 18-24 months in rats. 244 However, a 15-year postmarketing surveillance study shows that teriparatide does not increase adult osteosarcoma, advising to remove the black box warning regarding the potential risk of osteosarcoma. 245,246 This may be because the 2-year treatment runs through the whole life of rats while only occupying a small part of human life. Furthermore, daily subcutaneous injection administration may be one of the reasons for poor patient compliance. Therefore, a series of experiments on oral delivery of PTH or long-acting and slow-released preparation is being carried out. Including a weekly dose teriparatide encapsulated dissolving microneedle patch, 247 an orally available enteric-microencapsulated teriparatide-deoxycholic acid nanocomplex, 248 a triple padlock nanocarrier prepared by a taurocholic acid-conjugated chondroitin sulfate, 249 and an oleic acid-based dispersion in combination with chitosan-teriparatide polyelectrolyte complex, 250 whose efficacy needs further verification.
Abaloparatide is a second-generation osteoanabolic drug that was only approved in the USA in 2017. For now, teriparatide and abaloparatide are used to treat osteoporosis at high risk for fracture or failure or intolerance to other available osteoporosis therapies, and they are not recommended to be used for more than 2 years. 251,252

RANKL-binding peptide
OP3-4 is an OPG-like peptidomimetic that shows promising effects in reducing bone loss in OVX mice. 253 In the mouse tooth extraction model, local application of OP3-4 can decrease the number of osteoclasts and induce new bone formation. 254 At the same time, OP3-4 is injected under the jaw periosteum of normal mice, and the new bone mineralization proceeds from outside to inside, indicating that OP3-4 may participate in the early steps of accelerating osteogenesis. 255 WP9QY is structurally similar to the cysteine-rich domain of type I TNF receptor. It blocks the downstream signal transduction of RANK by inducing the conformational change of the RANK extracellular domain 256 ( Figure 4A). Data from in vitro experiments indicate that WP9QY can enhance the osteogenic differentiation of MSCs, stimulate the proliferation of osteoblasts and promote the apoptosis of osteoclasts. [257][258][259] In OPG-/mice, WP9QY suppresses osteoclastogenesis by inhibiting RANKL and enhances osteoblastogenesis by attenuating sclerostin expression in the alveolar bone. 260 The cyclic peptide L3-3 strongly binds to Loop3 of ectodomain RANKL and blocks RANKL-induced osteoclast differentiation more efficiently than OP3-4. 261 L3-3B further reduces the length of the peptide chain based on L3-3. In vitro experiments show that L3-3B can interfere with the phosphorylation of p38 and AKT in bone marrow-derived macrophages induced by RANKL. 262 Unfortunately, there is no experiment using L3-3 in vivo.

Alternative options
At present, the pharmacological options for osteoporosis are mainly achieved by inhibiting bone absorption or promoting bone formation. However, long-term or highfrequency use of these drugs may bring some side effects and adverse reactions. Therefore, further researches focus on the evaluation of the dose and route of administration and combination medication. Meanwhile, progress in studies about the molecular mechanism of maintaining bone homeostasis offers opportunities for new medications, and more effective therapies on specific targets remain to be further developed. On the other hand, biological therapy via stem cells and their secretome has also received broad attention. In the following part, we will summarize the new therapeutic ideas, hoping to give some alternative options.

Polypharmacy in osteoporosis treatment
Although the strength of the evidence may vary, almost all the drugs have been demonstrated for reducing vertebral and nonvertebral fracture risk. 24 The selection of osteoporosis treatment should be individualized and consider a variety of factors. 263 Among them, the combination of drugs with different mechanisms may bring additional benefits. 264 Furthermore, the sequential use of drugs can avoid the increased risk of specific side effects and minimize the resistance caused by the long-term use of a single drug. 265 However, it is not recommended to use two anti-absorbent agents at the same time because of the excessive inhibition of bone turnover and increased risk of fracture. 266 Anabolic therapy first, followed by potent antiresorptive therapy is an ideal plan. 223,267 The benefit-risk profile of Bps or denosumab is likely to be favorable for up to 10 years in individuals at high fracture risk. Anabolic therapy for up to 12-24 months should be considered for those at high or imminent fracture risk, followed by an antiresorptive drug. 268 But owing to economic and compliance reasons, the majority of anabolic therapies are based on dissatisfaction with previous anti-absorption therapies, most of which had taken Bps for several years. 269 Unfortunately, both Bps and denosumab will show progressive or transient bone loss when switching to teriparatide. 267, 270 An overlap of 6-12 months of PTH is recommended instead of stopping Bps immediately when switching from Bps to prevent the transient loss of BMD in cortical sites. 271 The combination of PTH and SERMs or denosumab appears to show continuous beneficial profits. 272,273 But it is worth noting that the combination of Bps and PTH would not bring additional benefit to BMD. In contrast, Bps may reduce the anabolic effects of PTH. 274 Recently, Jörg et al. 275 developed a digital framework that can predict the effects of drugs used to treat PMOP. This framework unifies the fundamental mechanisms of Bps, PTH, RANKL inhibitors, and sclerostin inhibitors. This model suggests that medication schemes eliciting a rapid BMD increase may not continuously improve BMD, cautioning that long-term indicators such as fracture should be taken as the measurement standard instead of BMD only. 275

Stem cell-based therapy
Due to the fear of rare side effects and concerns regarding long-term efficacy, the existing anabolic agents are not as well applied. Stem cell-based regenerative therapy is considered as a new approach to regenerating bone tissue. Among them, MSCs have attracted much attention due to their lower immunogenicity, higher proliferation, differentiation potential, and extensive access. 276 In addition to immunomodulation and differentiation potential, MSCs also have a strong paracrine effect. 277 Therefore, EVs containing paracrine factors, which are isolated from MSC conditioned medium, offer a strategy for cell-free MSC therapy. 278,279 Several studies have shown that human-derived MSCs can enhance osteogenesis and prevent OVX-mediated bone loss in mice, [280][281][282] but the specific mechanism is unclear. Wang et al. 283  maintaining estrogen levels to mediate adipogenesis and osteoblastic metabolism in bone. Through femoral artery ligation, Wang et al. 284 found that BMSC transplantation can promote the establishment of collateral circulation and intraosseous microcirculation at the ischemic sites, thereby inducing osteoporosis formation in OVX mice. Mei et al. 285 proposed a novel method to screen MSCs with high migration capacity, which enhances PDGFR/Wnt/βcatenin activity, forms more bone nodules, and partly rescues the bone loss of OVX rats. So far, stem cell-based therapy has mainly concentrated on animal experiments because of the uncertainty regarding the post-transplantation fate of stem cells and their safety in recipients. 284 Here, we summarize several clinical trials using stem cells to treat osteoporosis, none of which has entered phase III clinical trials ( Table 2).

DISCUSSION
Numerous efforts have been made to deal with osteoporosis. Nevertheless, the etiologies of osteoporosis involve a series of complex mechanisms, including osteoblasts, osteoclasts, cytokines, mechanical levers, and stem cells.
In this review, we narratively demonstrate the etiologies and underlying mechanisms of osteoporosis to provide readers with inspiration about new therapies. An example of a theory-medicine transformation is denosumab, and it is believed that the discovery of denosumab is a milestone in osteoporosis treatment. The approach to developing novel therapeutics shifted from observational or accidental findings to mechanism-based findings. 286 Besides, we also systematically review drugs in osteoporosis treatment, including therapies and concepts, which are newly developed. We believe that these works will benefit clinical workers or pharmaceutical workers in the future. Through our collection, we realize that there are still enigmas in the pathogenesis of osteoporosis and that drugs based on current theoretical mechanisms may not be beneficial. One of the examples is the discovery of cathepsin K, which is the function substance of osteoclast resorption. 287 The cathepsin K inhibitor odanacatib inhibits bone resorption significantly and has once been considered a promising treatment for osteoporosis. But a multicentral, randomized, double-blind, placebo-controlled trial revealed an increased risk of cardiovascular events in the odanacatib group, resulting in its development discontinuing. 288 The mechanisms related to its adverse events remain incomprehensive.
In clinical applications, individualized medication regimens should be taken into account. Osteoporosis is more likely to occur in older age groups with multiple comorbidities. Fracture risk rises in older adults with high levels of high-density lipoprotein cholestrol. 289 Clinical studies have shown that in obese people, the response to vitamin D is blunter. 290 In summary, more attention should be paid in groups with systematic disease and individual therapy should be designed. Unfortunately, older adults with multiple comorbidities were typically excluded from the pivotal osteoporosis RCTs, which led to insufficient knowledge about drug therapy in patients with systemic disease.
Furthermore, it may be another way to develop by shifting the focus from using drugs to inhibit bone resorption to promoting bone synthesis or combining therapies. Studies have shown that anabolic agents have greater antifracture efficacy and produce larger increases in bone density than anti-resorptive drugs, but anabolic drugs have a shorter effective time, so the transition to anti-resorptive drugs is required. 291 Further investigations are needed to fully understand the mechanisms and cross-talks of osteoporosis, in order to develop new strategies or moderate existing strategies to counteract osteoporosis and enhance the quality of life of patients.
Generally speaking, advances in the mechanisms of osteoporosis have shed light on the limitations and crises of current treatments. The uncertain and controversial insights in mechanism make the development of new approaches with high efficiency and few side effects remind challengeable. In addition, we also need to go beyond the limitations of mainstream therapies and use more personalized therapies according to the patient's etiology, lifestyle, and complications.

A U T H O R C O N T R I B U T I O N S
F.Y. conceived the study. H.W., Y.C.L., F.L., and H.S.W. performed literature searching and summary. H.W., Y.C.L., F.L., and F.Y. wrote the manuscript. F.Y., F.L., and L.Y. edited the manuscript. All authors have read and approved the final manuscript.

A C K N O W L E D G M E N T S
This work was supported by National Natural Science Foundation of China (U21A20368 to L.Y., 82100982 to F.L., and 82201045 to F.Y.); Sichuan Province Science and Technology Program (2021JDRC0144 to F.L. and 2022JDRC0130 to F.Y.); and Young Elite Scientist Sponsorship Program by CAST 2022QNRC001 (F.Y.). All figures in this review are created in Biorender.com.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All data needed to evaluate the conclusions in the paper are present in the paper.

E T H I C S S TAT E M E N T
Ethics approval and consent to participate not applicable.